Adenylate cyclase is a membrane-bound enzyme which plays a key role in the regulation of cellular function and the mediation of hormonal action. Although significant knowledge of the properties of this enzyme and its relationship to hormone receptors has been forthcomming, a clear understanding of its regulation and the mechanism of its linkage to the receptor in the membrane as well as co-factors critical for its activity can only come following its purification and subsequent characterization. Present attempts to purify this enzyme in our laboratory have included the use of hydrophobic chromatography to separate the enzyme from other intrinsic membrane proteins. Following this step, cyclase can be shown to display a more typical hydrophilic character and the detergent is no longer required to prevent its aggregation. Following this technique, we are planning to use affinity purification on nucleotide-agarose resins to obtain large-scale purifications of the enzyme. Furthermore, attempts at raising antibodies to specific subunits of the enzyme are being attempted. Specifically, following cholera intoxication, it has been shown that an ADP-ribose moiety is added to the GTP binding subunit of adenylate cyclase. Utilizing antibodies to the ADPR moiety, it is hoped that we will be able to specifically separate this subunit as well as achieve a significant degree of purification. Finally, we have also been able to separate out the GTP subunit using previously published techniques and can reconstitute the adenylate cyclase system by the addition of the GTP binding subunit to the isolated catalytic unit. At the present time, the Milstein monoclonal technique is being employed in an attempt to raise monospecific antibodies to each of these subunits. In the long term, it is hoped that we will be able to directly characterize the purified enzyme including determination of its stokes radius, partial specific volume, molecular weight and symmetry. We also intend to develop a radioimmunoassay for the enzyme so that we can directly address the question of how a cardiac cell membrane is modified during states of hypertrophy. Of significant interest will also be attempts to probe its hydrophobic binding site and to obtain a picture of just how the enzyme is anchored to the membrane.